Bottom Line:
Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

ABSTRACTThe Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.

Figure 3: PLOD-1 and Plod-2 promoter elements bind PITX2 in vitro. (A) EMSA of PITX2 protein incubated with radioactively labeled double stranded oligodeoxyribonucleotide probes designed from the PLOD-1 promoter. Bic, Drosophila bicoid element. For sequences of Bic and of elements C, H, and J see Fig. 2 and Table . (B) EMSA of PITX2 protein incubated with probes designed from the Plod-2 promoter. For sequences of elements C, E, F, and G see Fig. 2 and Table .

Mentions:
We screened a mouse genomic library and retrieved a BAC clone containing the Plod-2 gene. We sequenced 2,551 bp upstream of where the 5′ end of the published cDNA sequence starts. Here we discovered several sites shown previously to bind Drosophila bicoid specifically by DNaseI footprinting (Fig. 2). Sites A and C resemble the “X3” site, and site D resembles the “X1/X2” sites (Ma et al. 1996). Site F resembles the “B1” site (Driever and Nüsslein-Volhard 1989). Other similar sites have been shown to bind bicoid specifically (Rivera-Pomar et al. 1995; Dave et al. 2000). We confirmed that the C, E, F, and G sites also bound PITX2 by EMSA (Fig. 3 B). Negative controls were performed for all probes (probe only), but in Fig. 3 B only the control for the C probe is shown.

Figure 3: PLOD-1 and Plod-2 promoter elements bind PITX2 in vitro. (A) EMSA of PITX2 protein incubated with radioactively labeled double stranded oligodeoxyribonucleotide probes designed from the PLOD-1 promoter. Bic, Drosophila bicoid element. For sequences of Bic and of elements C, H, and J see Fig. 2 and Table . (B) EMSA of PITX2 protein incubated with probes designed from the Plod-2 promoter. For sequences of elements C, E, F, and G see Fig. 2 and Table .

Mentions:
We screened a mouse genomic library and retrieved a BAC clone containing the Plod-2 gene. We sequenced 2,551 bp upstream of where the 5′ end of the published cDNA sequence starts. Here we discovered several sites shown previously to bind Drosophila bicoid specifically by DNaseI footprinting (Fig. 2). Sites A and C resemble the “X3” site, and site D resembles the “X1/X2” sites (Ma et al. 1996). Site F resembles the “B1” site (Driever and Nüsslein-Volhard 1989). Other similar sites have been shown to bind bicoid specifically (Rivera-Pomar et al. 1995; Dave et al. 2000). We confirmed that the C, E, F, and G sites also bound PITX2 by EMSA (Fig. 3 B). Negative controls were performed for all probes (probe only), but in Fig. 3 B only the control for the C probe is shown.

Bottom Line:
Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

ABSTRACTThe Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.